Yishen Tongluo formula regulates apoptosis and autophagy in testicular spermatogenic cells of oligoasthenozoospermic rats via the AMPK/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Yishen Tongluo Formula (YSTLF) is a modern compound formula derived from the traditional Chinese medicine (TCM) theory of "kidney deficiency and collateral obstruction", and consists of seven Chinese medicinal herbs. It has been used clinically to treat oligoasthenozoospermia (OAS) for more than two decades, with proven efficacy and a favorable safety profile. However, its precise molecular mechanism has yet to be fully elucidated.
AIM OF THE STUDY: The aim was to investigate the therapeutic efficacy and the underlying mechanism of YSTLF in treating OAS associated with the kidney deficiency and collateral obstruction pattern.
MATERIALS AND METHODS: The chemical constituents of YSTLF were identified by UHPLC-Q-Orbitrap HRMS, and molecular docking was performed between the ten most abundant compounds and AMPK/mTOR. Rat OAS models were established by combined GTW, adrenaline, and ice-water immersion. Pharmacodynamic effects were evaluated through general status, reproductive organ weights, sperm quality, coagulation parameters, and serum sex hormones. Testicular histopathology, ultrastructure, and autolysosomes were examined by HE staining and TEM. Apoptosis was detected by TUNEL staining, and autophagy/apoptosis-related protein expression by IHC. Integrated metabolomics and proteomics with KEGG enrichment were conducted. In GTW-injured GC-1spg cells treated with Compound C or rapamycin, CCK-8 assay assessed viability, flow cytometry evaluated apoptosis, immunofluorescence examined LC3B/cleaved caspase-3 expression and colocalization, and Western blotting/RT-qPCR validated AMPK/mTOR pathway key molecules.
RESULTS: Among 307 identified compounds (mostly flavonoids), YSTLF improved general status, organ weights, and sperm quality in model rats. The ten most abundant compounds showed binding energies < -7.0 kcal/mol for AMPK/mTOR. Sperm concentration and motility increased dose-dependently. PT and TT were prolonged, FIB decreased, and FSH, LH, T, PRL, and Inh-B were restored. H&E, TEM, and TUNEL revealed alleviated testicular pathology, improved ultrastructure, reduced autolysosomes, and decreased apoptosis. IHC showed downregulated Beclin1 and Bax, and upregulated p62 and Bcl-2. Multi-omics revealed co-enrichment of differential proteins/metabolites in autophagy and mTOR pathways. Western blot/RT-qPCR validated downregulated p-AMPK/AMPK, AMPK mRNA, LC3II, Beclin1, Bax, cleaved caspase-3, and cleaved caspase-3/caspase-3 ratio, alongside upregulated p-mTOR/mTOR, mTOR mRNA, p62, and Bcl-2. In vitro, GTW reduced viability concentration-/time-dependently (stable model: 10%/24 h); YSTLF reversed this (optimal: 15%/24 h), restored viability, reduced apoptosis, and decreased LC3B/cleaved caspase-3 fluorescence/colocalization, while downregulating p-AMPK, LC3II, Bax and upregulating p-mTOR, p62, Bcl-2. Compound C alone mimicked YSTLF's effects without additivity, whereas rapamycin aggravated GTW-induced viability loss, apoptosis, and autophagy/apoptosis co-activation.
CONCLUSION: YSTLF improved spermatogenic function in rats with OAS (kidney deficiency and collateral obstruction pattern). Its mechanism of action involves regulation of the AMPK/mTOR signaling pathway and restoration of the balance between autophagy and apoptosis.